Agriculture resource guide Innovation. Applied.

Thermo Fisher Scientific is committed to providing instruments, reagents, and solutions for plant and animal research applications that will lead the way to remarkable agricultural discoveries—everything from improved crops to livestock that feed more people. We offer simple, scalable, and affordable solutions to help bring the next generation of agricultural products to life.

2 Thermo Fisher Scientific | Agricultural resource guide Contents

Overview of agricultural applications 4

Plant and animal de novo genome sequencing 5

Plant and animal genotyping 7

QTL mapping and marker-assisted selection 12

Plant and animal gene expression analysis 15

Synthetic biology and genetic engineering 16

Plant cell and tissue analysis 18

GMO testing and detection 20

Sample preparation and nucleic acid isolation 22

thermofisher.com/agbio 3 Overview of agricultural applications This application resource guide will help you select our best products for some of the most common agricultural science applications.

QuantStudio Genetic family of Ion Torrent TaqMan analyzers real-time PCR sequencers Assays systems

QuantStudio QTL mapping & family of marker-assisted Ion Torrent real-time PCR selection sequencers systems

Plant and animal TaqMan Genetic GMO testing de novo genome Assays and detection analyzers Objectives sequencing Higher yields Pest and environmental stress tolerance Healthier diets SimpliAmp GeneArt Thermal Cycler Precision TALs More efficient fertilizer use Higher quality meat Higher accuracy breeding values Phire QuantStudio Synthetic biology Higher feed conversion Plant and animal QuantStudio Direct PCR family of and genetic kits and real-time PCR genotyping Genetic family of master mixes systems engineering analyzers real-time PCR systems

Invitrogen GeneArt Gene custom DNA Plant and animal Synthesis Ion Torrent TaqMan oligos gene expression sequencers Assays analysis

GeneArt DNA Competent Ion assembly kits cells AmpliSeq for GBS Ion Torrent TaqMan sequencers Assays

PCR Gateway enzymes cloning

Qubit Fluorometer E-Gel with NanoDrop agarose gels Spectropho- tometer

GeneArt CRISPR products and services

4 Thermo Fisher Scientific | Agricultural resource guide Plant and animal de novo genome sequencing Genome sequencing allows researchers to Ion S5 System uncover the genetic makeup of plants and animals. Sequencing plant and animal genomes is becoming easier and more cost-effective—this is true even for de novo sequencing projects aimed at obtaining the primary genetic sequence of your species of interest. We have an extensive portfolio of sequencing instruments, reagents, and analysis software to help get you there faster and with greater accuracy and reliability.

Ion Chef System

De novo sequencing solutions The Ion S5™ and Ion S5™ XL systems provide the simplest DNA-to-data workflow for targeted sequencing with industry- leading speed and affordability. So you can spend less time doing repetitive lab work and more time answering the critical questions in your research. These dramatic improvements in sequencing technology are changing the way agricultural scientists look at genomics and are paving the way for the next wave of remarkable agricultural discoveries. Ion Chef System templating made easy Genome sequencing for all The Ion Chef™ System makes next-generation sequencing Fast and affordable genome, exome, and transcriptome more accessible to agricultural scientists across the sequencing of plants and animals is accessible with the globe, offering: semiconductor sequencing technology in the Ion S5 systems. It empowers researchers to sequence his or • Automated template preparation and chip loading her economically important species, large or small. The • Cartridge-based reagent system system offers: • Sample- and reagent-tracking capability • Intuitive user interface • Chips with 60–80 million reads—ideal for exomes, • Application flexibility with 35–400 bp sequencing transcriptomes, and genotyping by sequencing • Onboard diagnostics • High accuracy • 2–4 hour sequencing run time Find out more at thermofisher.com/ionchef • Single-day sequencing of plant and animal genomes

Find out more at thermofisher.com/ions5

Find out more and download case studies and technical information at thermofisher.com/plantdenovo

thermofisher.com/agbio 5 6 Thermo Fisher Scientific | Agricultural resource guide Plant and animal genotyping

The discovery of novel single-nucleotide polymorphisms (SNPs) is critical for plant and animal genotyping, as they serve as candidate markers for marker-assisted selection programs. Recent improvements in real-time PCR and next-generation sequencing have simplified SNP discovery and made it much more cost-effective. We offer a range of solutions to streamline all of your resequencing investigations and empower you to achieve your experimental and production goals.

Why are SNPs important? Targeted resequencing and SNP analysis solutions SNPs are small variations in the DNA sequences that are Ion Torrent™ sequencers for targeted resequencing enable associated with desired traits. They are used in conjunction you to discover new SNPs in important crop genomes. with quantitative trait locus (QTL) mapping to develop assays Confirm your discoveries either with Applied Biosystems™ for marker-assisted selection programs aimed at improving TaqMan™ Assays or by Sanger sequencing on one of the or enhancing certain characteristics such as higher yield, Applied Biosystems™ genetic analyzers. better stress tolerance, or enhanced pest resistance.

The table below compares the best genotyping solutions across our portfolio. Best experimental Research need Our platforms Why approach Compare sequences of Genotyping by Ion S5 and Ion Proton™ Speed, cost, scalability, simplicity several crop variants to sequencing (GBS) systems Ion Torrent™ systems enable fast discover functional SNPs and affordable GBS applications Map up to 10 SNPs in Fragment analysis SNP genotyping using Multiplexing capability: up to 10 different regions of the capillary electrophoresis (CE) SNPs per reaction genome genetic analyzers Map SNPs in a small region Sanger sequencing Sanger sequencing using CE Gold-standard technology for of the genome genetic analyzers sequencing Accuracy, low cost Simple workflow Confirm putative SNPs and Genotyping by Applied Biosystems™ Easy and fast workflow develop SNP assays for real-time PCR or TaqMan™ Sample-to-SNP™ High call rate and accuracy downstream work Ion AmpliSeq™ Kit Different formats for different technology Custom Applied project sizes (number of SNPs Biosystems™ TaqMan™ SNP combined with number of samples) Genotyping Assays and master mixes Real-time PCR instruments Ion Torrent™ sequencers Ion AmpliSeq™ consumables

Find out more at thermofisher.com/gbs

thermofisher.com/agbio 7 Plant and animal genotyping by sequencing Scalability, simplicity, and speed

Genotyping by sequencing Two strategies for genotyping by sequencing Whether it’s used for the discovery and identification of The Ion S5 and Ion S5 XL next-generation sequencing SNPs or as a screen for panels of thousands of known systems support two different GBS strategies: markers, GBS using NGS technologies is a cost-effective • Ion AmpliSeq™ products for GBS: In cases where a set and unique tool for association studies and genomics- of SNPs has been defined for a section of the genome, assisted breeding in a range of plant and animal species— this approach uses PCR primers designed to amplify the including those with complex genomes that lack a reference targets of interest. View our complete solution for multiplex sequence. Ion Torrent sequencers are useful tools for GBS PCR below. Creating and ordering custom panels is easy due to their scalability, simplicity, and speed. with the Ion AmpliSeq™ Designer—a free, online assay design tool that enables custom design for any genome. Advantages of genotyping by sequencing with Ion S5 Use the preloaded bovine, chicken, pig, sheep, maize, and Ion S5 XL next-generation sequencing systems: tomato, soybean, and rice genomes to design your • Allow both SNP marker confirmation and de novo custom panel, or easily upload a FASTA file with your SNP marker discovery, even in cases where there is desired reference sequences for your genome no reference genome of interest. • Provide attractive, low-cost options for large numbers • Restriction enzyme (RE) digestion: The complexity of of samples and data points the genome is reduced by digesting the DNA with one • Utilize highly multiplexed sequencing to scale or two selected restriction enzymes prior to the ligation from thousands to millions of data points per day of sequencing adapters. Compared to the multiplex PCR • Enable 2–4 hour sequencing run times to approach, which is used for targeting known variants, this facilitate efficient planning for the next reproduction cycle method is better suited for situations where little genome sequence is available or no prior SNP variants have been • Offer a high degree of accuracy of SNP calling identified, for discovering new markers for marker-assisted • Offer versatility for many applications, including selection programs. de novo sequencing, targeted resequencing, and transcriptome sequencing

Day 1 Day 2

1 DNA preparation 2 RE digestion/ 3 Ligate barcoded 4 Amplify library 5 Prepare template 6 Sequence 7 Analyze data multiplex PCR adapters fragments

A. SNP verification: A. Ligation of amplification of targeted sequencing adapters For the multiplex PCR regions with custom (specific to common approach, this step designed primer sets anchor sequences from is optional. and incorporated step 2). We offer an extensive In the RE digestion The Ion Chef System The Ion S5 Sequencer Ion Torrent Suite barcodes. portfolio of DNA B. Ligation of method, the selected for template preparation is ideal for GBS analysis software is isolation kits. B. SNP discovery: sequencing adapters library fragments are is automated. applications. used for SNP calls. gDNA restriction specific to restriction amplified using primer enzyme digestion. site; adapters contain sequences common to barcode sequence. the restriction sites.

8 Thermo Fisher Scientific | Agricultural resource guide Plant and animal genotyping by sequencing with Design for any genome Ion AmpliSeq technology Design and order custom panels with the Ion AmpliSeq Ion AmpliSeq technology uses ultrahigh-multiplex PCR Designer—a powerful but simple and easy-to-use web- to offer simple and fast library construction for targeted based tool. This free assay design portal connects you sequencing of specific genes or genomic regions. Custom directly to our assay design pipeline. Select your targets of primers are generated with high specificity using proprietary interest or enter your specific genomic content online, and software to target just your sequences of interest—up 5,000 we will design and create a customized Ion AmpliSeq™ panel amplicons in one reaction. just for you. Panels can be designed against a number of preloaded genomes (see below), or for any other organism Access our Ion AmpliSeq™ webinar on a tomato by uploading your own private reference sequence. Private targeted resequencing and SNP analysis study at references are stored securely using 128-bit SSL encryption. thermofisher.com/plantwebinars Simple, secure, and powerful—you will be able to design for any custom reference genome. Visit ampliseq.com and start creating your designs today.

Rice Cow Tomato (rice_v7) (bosTau7) (SL2.50)

Sheep Chicken Custom (oviAri3) (galGal4) Reference

Pig Maize Soybean (susScr3) (Zea_mays.AGPv3) (Glyma1.1)

Several preloaded genomes are available, or use the option to upload a custom reference sequence through the Ion AmpliSeq Designer.

thermofisher.com/agbio 9 Plant and animal genotyping by qPCR

Genotyping by qPCR Genotyping by real-time PCR—whether for the confirmation of SNPs or as a screen for panels of markers in hundreds or even millions of samples—is a well-known and demonstrated approach used widely in both research and industrial settings. We are a leader in providing real-time PCR instruments, TaqMan Assays, and reagents that are designed Applied Biosystems TaqMan Sample-to-SNP Kit to generate reliable results for validation and screening. This kit contains DNA Extract All Lysis Reagents that provide PCR-ready DNA from a wide variety of plant tissues, typically in 5 minutes. The kit also contains the Applied Biosystems™ TaqMan™ GTXpress™ Master Mix for robust PCR amplification of extracted DNA typically in less than 50 minutes.

Custom TaqMan SNP Genotyping Assays These assays provide a highly flexible technology for detection of polymorphisms within any genome. Create your own assays by submitting target sequences to our secure assay design pipeline using the Applied Biosystems™ Custom TaqMan™ Assay Design Tool. This pipeline has successfully generated millions of assay designs by utilizing heuristic rules deduced from both manufacturing and assay performance data. Applied Biosystems QuantStudio 12K Flex Real-Time PCR System Custom Applied Biosystems TaqMan Assay • Flexibility—five interchangeable blocks to fit your needs manufacturing and plating service • 110,000 genotypes per workday without robotics; other Whether it’s zygosity analysis or special manufacturing instruments are available to meet your throughput needs modifications such as a specific dye, assay volume or and budgets concentration, or plating format, we can tailor a service to meet your needs.

Overview of workflow for genotyping by real-time PCR

DNA isolation Reaction preparation Amplification Endpoint reading Analysis TaqMan Sample-to-SNP Assays are available in Perform PCR using the Read amplification with Applied Biosystems™ Kit provides PCR-ready different formats: single QuantStudio systems the Applied Biosystems™ TaqMan™ Genotyper DNA from a wide variety of tube, 96- and 384- or Applied QuantStudio™ 12K Software is a great sample types of plant and well plates, TaqMan™ Biosystems™ Flex Real-Time resource for fast and animal tissues, typically in Low Density Array, or Veriti™ Thermal PCR System accurate genotype 5 minutes OpenArray™ plate Cycler calling; TaqMan Mix your DNA sample Genotyper Software Amplification and endpoint reading with the Custom TaqMan can be downloaded at Can be performed with the GTXpress Master Mix thermofisher.com/ QuantStudio 12K Flex system and the TaqMan SNP taqmangenotyper Genotyping Assay

Together, the Custom TaqMan SNP Genotyping Assays and QuantStudio 12K Flex system provide the flexibility and throughput you need for your project. Contact your sales representative to discuss your needs.

Learn more at thermofisher.com/agbio

10 Thermo Fisher Scientific | Agricultural resource guide Results from the Delta Genomics bovine Results from the International Rice Research Institute Ion AmpliSeq panel (IRRI) rice Ion AmpliSeq panel Delta Genomics is a national, not-for-profit provider of IRRI, located in Los Baños, Philippines, aims to reduce genomics services including genotyping, sequencing, poverty and hunger, improve the health of rice farmers and and biobanking for the livestock industry and research consumers, and ensure environmental sustainability of community. The Delta Genomics cattle parentage Ion rice farming. The IRRI Ion AmpliSeq panel targets 500 rice AmpliSeq panel targets 121 markers across a total of 14,153 genetic markers across a total of 84,572 bp in a single bp in a single primer pool. The average call rate was >95% primer pool. The average call rate observed was >94% and the average genotype concordance was >98% for 104 for the panel when tested on validation samples. Using 56 positions in a comparison between the Delta Genomics Ion samples, the average genotype concordance was >98.9% AmpliSeq™ panel and bead-based microarray results for 160 for 299 positions in common between the IRRI Ion AmpliSeq cattle samples. panel and Illumina™ GoldenGate™ microarray data.

thermofisher.com/agbio 11 QTL mapping and marker-assisted selection

Effective, high-throughput genotyping methods for marker-assisted selection (MAS) are enhancing our understanding of how to breed crops with desired traits such as higher yields or better disease resistance. We offer a wide range of technologies to facilitate your breeding and functional genomic studies, associate important traits with genetic variants (including SNPs and single sequence repeats, or SSRs), construct QTL maps, and identify genetic markers for MAS programs.

What are QTL mapping and marker-assisted selection? What is the best approach if I am using SNP markers QTL analysis links genetic markers with DNA base variations, for breeding? such as SNPs and microsatellites or SSRs, to the QTLs Choosing the best experimental approach and technology of interest for breeding purposes. These are then used in platform depends on your specific needs. Two key elements linkage mapping and downstream marker-assisted breeding to consider are the number of samples and the number of programs to screen for individual plants that have the trait(s) markers in your project. The chart below gives a high-level of interest. Depending on the program, these traits could be representation of the different technologies we offer to assist increased yield, disease or stress resistance, or enhanced you in selecting the right plant seed line, moving a transgenic nutritional or oil profiles. plant to the greenhouse or the field, or collecting critical data for your publication.

5,000 GBS on Ion Proton and Ion S5 systems • Low cost for total project • Fast and easy workflow • Flexibility • Affordability TaqMan Assays on QuantStudio OpenArray platform 500 • Low cost • Proven Applied Biosystems™ TaqMan™ TaqMan Assays, high- chemistry resolution melt (HRM) • Streamlined workflow analysis, SSRs • Reduction in consumables and steps • Fast and easy workflow TaqMan Assays on Douglas Scientific Array Tape™ strips in a design or panel 100 • Gold standard with high call rate and accuracy • High throughput (TaqMan Assays) • Automated workflow • Very low-cost HRM analysis • Low cost Number of SNPs or other markers • Proven TaqMan chemistry 10 10 100 500 1,000 5,000 300,000

Number of samples screened in a design or panel

12 Thermo Fisher Scientific | Agricultural resource guide Fragment Genotyping by TaqMan HRM TaqMan TaqMan analysis sequencing Assays on analysis Assays on Assays on (SSRs) on Ion Torrent QuantStudio QuantStudio Douglas sequencers family of OpenArray Scientific real-time platform Array Tape PCR strips systems Sample 1–500 1 to millions 1–500 1–500 500–4,000 500 to millions throughput Marker 1–100 100–5,000 1–384 1–384 16–512 1–100 throughput Cost per Medium Low to medium Medium Very low Low Very low data point Formats and Multiplex up to Ion AmpliSeq Single tube, Single tube, OpenArray Array Tape flexibility 20 markers per Custom Panels 48-well, 96- 48-well, 96- plates plus strips sample (primer pools) well, 96-well well, 96-well four additional can be used to Fast, and Fast, and blocks for analyze up to 384-well 384-well different sample 5,000 amplicons in formats formats or marker 384 samples on a throughput single Ion PI™ Chip

DNA-to- 4 hr 1 day 40 min (Fast); 1 hr 4 hr 3 hr to 1 day answer time 2 hr (standard)

Download application notes and scientific articles highlighting these applications and learn more at thermofisher.com/qtl

thermofisher.com/agbio 13 14 Thermo Fisher Scientific | Agricultural resource guide Plant and animal gene expression analysis

Whole-transcriptome analysis is very important in understanding how altered expression of genetic variants contributes to complex plant and animal phenotypes. We offer an extensive product portfolio for gene expression analysis, from transcriptome sequencing and Applied Biosystems™ TaqMan™ real-time PCR solutions to products for epigenetics research—all aimed at facilitating gene expression analysis in a fast, reliable, and accurate way.

What are the different approaches for gene expression analysis? Agricultural researchers utilize a variety of approaches to understand gene expression. In some cases, whole- transcriptome profiling is preferred over targeted RNA sequencing, which generally involves panels. In order to study plant and animal functional genomics, researchers also utilize epigenetics tools as well as miRNA, small RNA, and RNAi studies to decipher the root cause behind phenotypes of interest. No matter what your study involves, we have a wide portfolio of instruments, reagents, and solutions for all Targeted gene expression analysis your gene expression studies. If your experiment is limited to a few genes or certain areas of the transcriptome, real-time PCR assays with Applied Transcriptome sequencing Biosystems™ TaqMan™ probes are the best approach for Compared to traditional microarray technology, which you. With trusted Applied Biosystems™ instruments and can only be used for gene expression profiling in species software, Applied Biosystems™ TaqMan™ predesigned and with known transcriptome sequences, NGS technologies custom gene expression assays and master mixes are enable agricultural researchers to perform these studies in tailored for success. The predesigned assays for soybean, any species—and to do so with assays that have a higher corn, rice, cattle, and pig allow you to spend your time dynamic range at a lower cost. If your goal is to profile the generating results, not on assay design and optimization. entire transcriptome or to use RNA sequencing to reduce Find out more at thermofisher.com/taqmangex the complexity of a large polyploid genome, transcriptome sequencing has never been easier than with the Ion S5 or Epigenetics, miRNA, and noncoding RNA research the Ion Proton System. Gene regulation in plants and animals occurs via epigenetic changes such as DNA methylation, changes in chromatin structure, and regulation by noncoding RNAs. These processes are part of a complex regulatory circuit that requires robust tools to study. We have developed powerful technologies for epigenetics research that span sample preparation, sequencing, quantitative PCR, functional analysis assays, and bioinformatics tools for analysis. View all our solutions at thermofisher.com/epigenetics

Browse all our solutions for gene expression at thermofisher.com/plantge Ion S5 System

thermofisher.com/agbio 15 Synthetic biology and genetic engineering

Generating transgenic plants is key to introducing new crop traits and carrying out gene studies, both in discovery and applied settings. The development of genetically modified plants often requires a complex design of DNA elements to achieve optimum effects on expression. We offer solutions for genome editing, cloning, DNA assembly, amplification, and analysis, as well as Agrobacterium tumefaciens LBA4404 for plant-cell transformation.

Manipulate, synthesize, and clone plant DNA easily Precise, efficient gene sequence targeting and The construction of T-DNA vectors or vectors for particle modification technologies bombardment or electroporation is often complex—and is From precision genome editing and gene modification a process that we’ve made easier through the development technologies to high-efficiency delivery systems, we have of innovative DNA cloning and assembly products. We developed a broad range of solutions to help you create understand the challenges of cloning and have the right the modified genes, expression systems, and stable cell products for you. Using our plant molecular biology solutions, lines you need for your research—from culturing cells to you can create multiple vectors to test the effect of regulatory modification, detection, and analysis. Our technologies— sequences in the target plant, or stack traits on the same including Invitrogen™ GeneArt™ CRISPRs and TALs—enable expression vector for crop enhancement. you to reliably modify genomic sequences and analyze the phenotypic outcomes. In addition, we can deploy a combination of advanced technologies to produce a custom construct or cell line that meets your specifications. Balancing performance and cost, our cell engineering portfolio is built on 20 years of innovation and can grow with your research needs.

GeneArt CRISPRs Introducing the only complete genome editing solution • Invitrogen™ GeneArt™ Seamless PLUS Cloning and designed to expedite your research. Our easy-to-use Assembly kits and GeneArt™ Gene Synthesis services: optimized and validated solutions make genome editing Our latest technology for simultaneous cloning of complex accessible to anyone at any level. We are continuing to DNA elements can be combined with GeneArt Gene expand our suite of genome editing products to span the Synthesis services for the design of synthetic genes with entire cell engineering workflow, from cell culture reagents, enhanced heterologous gene expression in transgenic delivery reagents, and sample preparation to genome plants. Learn more at thermofisher.com/geneart modification, and to detection and analysis of known genetic • Invitrogen™ Gateway™ cloning: The expression platform variants. We offer our state of the art online CRISPR search of choice for plant systems, with a wide range of vectors and design tool along with CRISPR-Cas9 in four formats, an for different plant species and purposes. Invitrogen™ all-in-one expression vector, Cas9 mRNA, Cas9 protein, and Gateway™ vectors adapted for Agrobacterium-mediated CRISPR Libraries services paired with the optimal cell culture transformation, silencing studies, and many other reagents, delivery method, and analysis tools based on your applications have been cited in over 150 peer-reviewed application and cell type. articles. Learn more at thermofisher.com/gateway • Basic cloning reagents: From competent cells to DNA oligos and Invitrogen™ TOPO™ cloning kits, we have everything you need for your everyday cloning needs. Browse our entire offering at thermofisher.com/cloning

16 Thermo Fisher Scientific | Agricultural resource guide GeneArt Precision TALs Plant transformation Based on Xanthomonas effector proteins, Invitrogen™ Agrobacterium-mediated transformation is the GeneArt™ Precision TALs are custom DNA-binding proteins easiest, simplest, and most widely used transformation that allow you to accurately target any locus in the genome. technique in plant biotechnology applications, and we TAL technology has been shown to be effective in a broad offer A. tumefaciens LBA4404 cells and antibiotics to range of organisms, including plants, algae, and fungi. enable your research in this field. GeneArt TALs are the first of their kind and are ideal for plant research scientists, offering research-use access with a clear Browse all our products for plant genetic engineering at licensing path for commercial use through the Two Blades thermofisher.com/plantengineering Foundation (2blades.org). Explore our precision TALs at thermofisher.com/tal Plant cell and tissue analysis

From studying the morphology of plant Rubisco localization in maize cells—cell walls, organelles, and other cellular leaf section. Rubisco was localized compartments—all the way to complex gene using a rabbit anti-rubisco antibody and visualized using the highly cross- regulation and expression studies, cell tissue adsorbed Invitrogen™ Alexa Fluor™ analysis techniques and tools are critical not 488 goat anti–rabbit IgG antibody only for basic plant research, but also for crop (Cat. No. A11034). The 2.0 μm maize improvement programs. Key techniques such as leaf section illustrates the immunolocalization of rubisco in the chloroplasts of the bundle sheath cells surrounding the immunohistochemistry have been routinely used to vascular bundles. The red fluorescence, localized to the label diverse plant tissues and organelles including mesophyll plastids, is due to background autofluorescence cell walls, vacuoles, chromatin, nuclei, nuclear of chlorophyll. Lignin appears dull green and is localized to the xylem of the vascular bundle; cutin appears bright green membranes, and chloroplasts. and is localized to the cuticle outside the epidermis. Image contributed by Todd Jones, DuPont. To empower your research in plant cell and tissue analysis, we have a broad portfolio of fluorescent Pectin associated with reagents, kits, and instruments specifically plasmodesmatal pit fields of designed with your needs in mind. kiwifruit cells. Pectin, a component of the cell wall matrix and the main constituent of the middle lamella that Invitrogen fluorescent dyes and probes forms between daughter cell walls, was Invitrogen™ reagents including Invitrogen™ Alexa Fluor™ tagged with an anti-pectin monoclonal dyes, Qdot™ nanocrystals, Click-iT™ detection assays, and antibody, JIM 5. The primary antibody was detected and ProLong™ Gold Antifade Reagents are used in a wide range visualized with Invitrogen™ Alexa Fluor™ 488 goat anti–rat of plants for a variety of research applications, including: IgG (Cat. No. A11006). The primary antibody was a gift from Dr. Paul Knox, University of Leeds, UK. Image contributed • Antibody labeling by Paul Sutherland, The Horticulture and Food Research • Immunohistochemistry Institute of New Zealand, Ltd., Mt. Albert Research Centre. • Fluorescence in situ hybridization • Organelle and cytoskeletal stains • Endocytosis studies • Transport studies • Glucose metabolism • Viability staining • Glutathione detection • Calcium and other ion-imaging applications • Tyramide signal amplification (TSA)

18 Thermo Fisher Scientific | Agricultural resource guide Flow cytometry Flow cytometry is an ideal means for the analysis of cells and subcellular particles, with a potentially large number of parameters analyzed rapidly, simultaneously, and quantitatively, thereby furnishing statistically exploitable data and facilitating accurate detection of subpopulations. Since the first report of its use with plants 40 years ago, flow cytometry has become an essential tool for the understanding of fundamental mechanisms and processes underlying plant growth, development, and function. Key applications include the study of ploidy levels, genome mapping, localization of sex-specific chromosomes, and plant cell and algal studies.

Find out more and see all of our products at A B All events Scatter thermofisher.com/plantimaging 106 106 Cell Imaging

Designed to eliminate the complexities of microscopy 105

without compromising performance, the Invitrogen™ 105

Side scatter Side (603/48) EVOS™ line of cell imaging systems makes cell imaging 104 accessible to almost every lab and budget. From cell culture fluorescence PI-DNA 103 104 to complex protein analysis to multichannel fluorescence 103 104 105 106 104 105 103 imaging, EVOS cell imaging systems help you perform a PI-DNA fluorescence (603/48) PI-DNA fluorescence (≥640) variety of routine and specialty applications. C Nuclei D Nuclei 4C 2C Cell Counting 600 4C 400 8C The new Invitrogen™ Countess™ II FL Automated Cell 500 16C 2C Counter is a benchtop assay platform equipped with state- 300 400 8C 300 of-the-art optics, full autofocus, and image analysis software Count 200 Count 200 for rapid assessment of cells in suspension. With three- 16C 100 100 channel flexibility—brightfield and two optional fluorescence 32C 0 0 channels—researchers can count cells, monitor fluorescent 104 105 106 50 100 150 200 250 300 protein expression, evaluate apoptosis, and measure PI-DNA fluorescence (603/48) PI-DNA fluorescence (603/48) cell viability. Nuclear holoploid genome sizing analysis of Arabidopsis thaliana leaf tissue homogenates using the Applied Biosytems™ Attune™ Flow Cytometer. (A) Biparametric density plot of side scatter vs. propidium iodide (PI)-DNA fluorescence, with scatter gate surrounding the fluorescent nuclei. (B) Biparametric density plot of PI fluorescence (603/48 vs. ≥640 nm) of scatter gate population from A. (C) Logarithmic histogram of PI- DNA fluorescence (603/48 nm) of nuclei gate population from B.(D) Linear histogram of PI fluorescence (603/48 nm) of nuclei gate population from B. 2C, 4C, 8C, 16C, and 32C denote the C-values for the respective peaks.

thermofisher.com/agbio 19 GMO testing and detection

Whether you are conducting genetically modified organism (GMO) testing to detect bioengineered GMOs in a sample for quality-control purposes, detecting allergens in food, or quantifying mutant and rare alleles in plant genomes, we offer a range of real-time and digital PCR solutions to address your throughput, accuracy, and detection sensitivity needs with high confidence.

Products and technologies for GMO testing Digital PCR solutions for GMO detection Testing for GMOs in food is now required in an increasing Digital PCR is a newer approach to nucleic acid detection number of countries and is becoming increasingly important and quantification that is ideal for accurately quantitating in quality-control tests. We offer kits that will allow you low amounts of plant transgenes in a wild-type background. to accurately detect GMO-specific DNA sequences in It doesn’t rely on reference standards and offers a high seed, grain, and processed foods and their ingredients. tolerance for multiple inhibitors of PCR that are encountered These GMO kits are based on TaqMan chemistry and in plant samples. are compatible with our wide selection of real-time PCR instruments. Find out more at thermofisher.com/digitalpcr

Explore our GMO testing and detection solutions at thermofisher.com/gmo

Applied Biosystems™ QuantStudio™ 3D Digital PCR System enables high accuracy, sensitivity, and absolute quantification

100%

Positive reactions Absolute quantification Sample partitioned Negative reactions into many reactions

20 Thermo Fisher Scientific | Agricultural resource guide Case study: Soybean (Glycine max) Dr. Wing Cheung (DNA LandMarks, BASF) partnered with us to design TaqMan Assays for GMO soybean DNA detection and to validate assay specificity with target-specific gDNA with a digital PCR approach. Using a 1:10,000 GMO/wild-type spike-in ratio, the authors confirmed that the measured ratio for GMO and wild-type targets was comparable to the estimated GMO/wild-type ratio.

Download the study at thermofisher.com/gmo Sample preparation and nucleic acid isolation

Plant DNA isolation has unique challenges that require reagents specifically designed to deal with carbohydrates, phenolics, and other compounds abundant in plant tissues. Plant cell walls can be very difficult to disrupt, and lysates often contain significant amounts of compounds such as tannins, phenolics, and complex polysaccharides that can affect DNA and RNA quality and inhibit downstream reactions. We offer an expansive portfolio of kits, reagents, and devices for the isolation, purification, analysis, and quantitation of not only plant genomic DNA but also total RNA, mRNA, and microRNA or transcriptome RNA.

Which DNA isolation kit is right for you? Bulk buffers for DNA extraction The most efficient Most suitable for Silica-based Magnetic beads crude extraction of and TaqMan lysis for the largest screening and method for low- designed to DNA and RNA from chemistry–based amount of tissue GMO testing due abundance plant handle low- plant cells genotyping to automation DNA samples abundance plant combined, <1 hr capabilities DNA samples to results Total RNA Lysis TaqMan Sample- Invitrogen™ Plant Invitrogen™ Invitrogen™ Invitrogen™ Solution to-SNP Kit DNAzol™ Reagent ChargeSwitch™ PureLink™ Genomic MagMAX™ DNA gDNA Plant Kit Plant DNA Multi-Sample Kit Purification Kit Top seller Yes Yes Yes

Starting material ≥100 mg 2–3 mm punch ≥100 mg 50–100 mg 100 mg 5–10 mg Yield Varies based on Varies based on Varies based on Up to 7 μg Up to 14.6 μg Up to 14.6 μg starting material starting material starting material

Protocol time <15 min 5 min <60 min <15 min <40 min <40 min Plant sample Customer-validated Broad range of Broad range of Some of the many Chloroplasts and Chloroplasts and types for DNA on a broad plant tissues and plant tissues (alfalfa species and tissues broad range of broad range of range of plant tissues starting material and sunflower are canola, corn, plant tissues (alfalfa plant tissues (alfalfa for SNP genotyping (leaf punches, sprouts, corn cotton, sunflower, and sunflower and sunflower as well as corn flour needles, and seed husks, soybeans, barley, rice (seeds), sprouts, corn sprouts, corn for GMO testing chips) mushrooms, tomato tomato, cucumber, husks, soybeans, husks, soybeans, leaves, wheat grass, lettuce, banana mushrooms, tomato mushrooms, tomato and Arabidopsis (leaves), rose, leaves, wheat grass, leaves, wheat grass, thaliana leaves) magnolia (flower), and Arabidopsis and Arabidopsis Acer, sycamore, and thaliana leaves) thaliana leaves) grass Isolation Bulk lysis buffer Lysis solution Organic extraction Invitrogen™ Silica spin column Magnetic beads technology ChargeSwitch™ chemistry, magnetic beads Downstream applications PCR, real-time PCR Real-time PCR All All All All

Automatable No Yes No Yes No Yes High throughput– Yes Yes Yes Yes No Yes compatible High Invitrogen™ throughput/ 50 preps Yes Yes Yes ChargeSwitch™ 96-well plate automation 96 preps gDNA Plant 960 Kit options Kit sizes 250 mL 100 preps 100 mL 96 preps 50 preps 50 preps available 400 preps 960 preps 4 x 96 preps 96 preps 4,000 preps

22 Thermo Fisher Scientific | Agricultural resource guide Which RNA isolation kit is right for you?

Great for difficult samples Rapid and fully Quick and easy to use Efficient recovery of (conifer tissue and seeds) automated microRNA and small RNA Plant RNA Reagent Invitrogen™ MagMAX™-96 Invitrogen™ PureLink™ Invitrogen™ mirVana™ miRNA Total RNA Isolation Kit RNA Mini Kit Isolation Kit Top seller Yes RNA types isolated Large RNA molecules only Large RNA molecules only Large RNA molecules only Small and large RNA molecules Prep time 60 min <45 min <20 min 30 min Amount of starting Up to 1 g Up to 10 mg <250 mg 0.5–250 mg material Isolation method High-purity organic extraction Scalable, flexible format Fast, convenient silica Highest purity and (requires alcohol precipitation) with magnetic beads column convenience; includes organic extraction and silica column (no precipitation required) High throughput– Yes compatible Compatible Invitrogen™ Plant RNA Plant RNA Plant RNA products Isolation Aid—removes Isolation Aid—removes Isolation Aid—removes common plant common plant common plant contami­nants contaminants contaminants

Explore our solutions at thermofisher.com/plantnap

thermofisher.com/agbio 23 Ion Torrent™ Capillary Technology platforms semiconductor electrophoresis Real-time PCR sequencing sequencing Applications Ion S5 System 3730 Series Genetic QuantStudio family of QuantStudio 12K Flex Analyzers real-time PCR systems Real-Time PCR System

De novo sequencing   Targeted resequencing   Confirmation of next- generation sequencing  results SNP discovery (whole-   genome) SNP confirmation    QTL mapping     Marker-assisted selection     Genotyping by sequencing  Linkage mapping   

DNA Variety identification     Microsatellite/SSR analysis   AFLP™ analysis  Mutation (SNP) screening   GMO testing   Digital PCR  Rare-allele detection  HRM analysis   Copy number variation     Methylation    ChIP-Seq  Whole-transcriptome  sequencing Targeted gene expression   Gene expression

RNA   confirmation miRNA expression   siRNA analysis   Protein expression   Protein

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For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. AFLP is a trademark of KeyGene N.V. DNAzol is a trademark of Molecular Research Center, Inc. Array Tape is a trademark of Douglas Scientific, LLC. Illumina and Golden Gate are trademarks of Illumina, Inc.CO37963 0116